Biological control of Botrytis cinerea and Sclerotinia sclerotiorum on kiwifruit
Botrytis cinerea and Sclerotinia sclerotiorum are the two most serious pathogens on kiwifruit in New Zealand. Because of the pesticide regulations in some of the countries to which New Zealand exports fruit, total protection from Botrytis stem end rot with current dicarboximide fungicides is not possible. The aim of this thesis was to investigate biological control measures for Botrytis stem end rot and Sclerotinia diseases of kiwifruit. More than 1000 microorganisms, isolated from the leaves and flowers of kiwifruit during spring and autumn, and selected from BCAs reported to be effective against B. cinerea and./or S. sclerotiorum, were tested in vitro for their antagonistic ability against B. cinerea and S. sclerotiorum. Successful antagonists were those that, in dual culture on agar plates, produced a zone of inhibition, an area of browning of the pathogens, or grew rapidly over the pathogens and inhibited their growth. The fifty most promising isolates from the initial screen were tested on fruit for their ability to reduce Botrytis and Sclerotinia fruit rots. Mature kiwifruit were artificallv wounded and dual inoculated with a spore suspension of one of the fifty test organisms and either a conidial suspension of B. cinerea or a mycelial suspension of S. sclerotiorum. Following 8-12 weeks incubation in a cool store, fruit were assessed for Botrytis or Sclerotinia induced rot. Isolates of Bacillus spp., Epicoccum purpurascens, Pseudomonas sp. and Trichoderma. spp. reduced, Botrytis fruit rot from 92% (inoculated control) to 0%.Isolates of Alternaria spp., pestalotia sp. and a non-sporulating isolate also reduced the number of fruit rotting to some extent. Similarly, isolates of Bacillus spp., E purpurascens and Trichoderma spp. reduce d Sclerotinia fruit rot from 100% (inoculated control) to 0%. Isolates of Alternaria spp., Myrothecium verrucaria and Pestalotia sp. were also successful at reducing the level of Sclerotinia fruit rot. It was considered undesirable if potential biological control agents (BCAs) were able to colonize kiwifruit that were to be marketed for human consumption. In order to determine if microorganisms, shown to be effective in preventing Botrytis or Sclerotinia fruit rot, were capable of themselves colonizing fruit, isolations were made from fruit dual inoculated with B. cinerea, S. sclerotiorum and/or one of several BCAs. Strains of the BCAs Bacillus spp., Pseudomonas sp. and E. purpurascens were not found to be saprophytic on fruit. Isolates of Alternaria sp., Bacillus sp., E purpurascens, pestalotia sp., Pseudomonas sp. and T. harzianum significantly inhibited germination and germ tube elongation of B. cinerea conidia in vitro in a nutrient solution, over a 24 h period. For example, the presence of Alternaria alternata A6 spores in a nutrient solution reduced germination of B. cinerea conidia from 100% to 20%. The presence of E purpurascens A77 spores inhibited B. cinerea conidial germ tube elongation from >840 pm (in control conidia) to 27 µm. The presence of any one of the BCAs tested prevented germination of B. cinerea conidia in a non-nutrient water solution, in comparision to germination of up to 86% in controls. A spore or cell suspension of each of the isolates Bacillus sp.M60, E. purpurascens A77 and T. harzianum C65 were spray inoculated onto kiwifruit blossoms produced in vivo in the glasshouse, immediately prior to inoculation of the blossoms with a condial suspension of B. cinerea. Application of the BCAs were completely effective in preventing colonization of blossoms by B- cinerea conidia. The effectiveness of each of the isolates E. purpurascens A77,T. harzianum C65 and either Bacillus sp.M60 or M53 to reduce the viability of sclerotia of B. cinerea and S. sclerotiorum was tested in soil punnets. A spore or cell suspension of each respective BCA was applied to the surface of replicated punnets that were seeded with either B. cinerea or S. sclerotiorum. Following 8 weeks incubation, punnets were harvested and viability of sclerotia assessed. T. harzianum C65 and Bacillus sp. M60 significantly reduced the viability of B. cinerea sclerotia from 8 sclerotia/punnet (control) to 4 sclerotia/punnet. T. harzianum C65 and E. purpurascens A77 caused a significant reduction in apothecia production of S. sclerotiorum, from 2.7 apothecia/punnet (control) to 0.7 apothecia/punnet. Bacillus sp.M8 and E purpurascens A77 were tested for their ability to reduce Botrytis stem end rot and Sclerotinia field rot in a kiwifruit orchard. The isolates tested did not successfully reduce either disease. Possible explanations for this are discussed. In order to monitor the survival of particular isolates of BCAs in the field, a technique was developed to distinguish between individual strains of a BCA species. The polymerase chain reaction (PCR) was utilized to identify DNA polymorphisms within the genome of T. harzianum C65, in comparison with other strains of Trichoderma spp.. A sequence of polymorphic DNA was cloned, sequenced and used as a hybridization probe in southern blotting to enable T. harzianum c65 to be distinguished from other strains of Trichoderma spp.. From the results obtained in this study, it was considered that Bacillus M60, E purpurascens 477 and Pseudomonas M30 were the best isolates for the biological control of Botrytis stem end rot on kiwifruit. Further work to enable application of these isolates as postharvest BCAs is discussed. Of the isolates tested in this study, T. harzianum C65 was considered the best isolate for use against Sclerotinia diseases on kiwifruit. Methods of selecting more effective BCAs against S. sclerotiorum are discussed.